The formation of septa and valves in the developing heart are crucial morphogenetic events that allow the initially single, embryonic cardiac tube to develop into a complex 4-chambered heart, supporting two different blood circulations. This development involves remodeling of the endocardial cushions, and (2) epicardialization, the migration of epicardially derived cells into the cushion tissues. Preliminary data indicate that perturbation of either process can lead to cardiac malformations involving the developing outflow tract (OFT) and atrioventricular (AV) function. The studies proposed in AIM 1 of this project are designed to address the overall working hypothesis that myocardialization is the "driving force" of segmental and septal alignment of the AV junction. The specific goals in this aim are (1) to elucidate the role of TGFbeta in the regulation of myocardialization and to test the hypothesis that perturbation TGFbeta signaling pathways in the outflow tract will lead to perturbation of myocardialization resulting in a specific set of congenital malformations including double outlet right ventricle (DORV) and double inlet left ventricle (DILV), and (2) to describe the myocardialization-induced remodeling of the linear curvature leading to the rightward expansion of the atrioventricular canal resulting in the connection between right atrium and right ventricle. The studies proposed in AIM 2 of this application focus on the role of epicardial derived cells (EPDCs) in valvuloseptal morphogenesis. The central working hypothesis here is that EPDCs play a crucial role in the regulation of mesenchymal cell transformation, proliferation, and differentiation of the endocardial cushions. The specific goals to be studied are (1) to test the hypothesis that proper development of the atrioventricular valves relies on timely regulated immigration of EPDCs into the endocardial cushion tissues, and (2) to test the hypothesis that EPDCs regulate endocardial cushion formation by local inhibition of endocardial-to-mesenchymal cell transformation and proliferation, and by promotion of differentiation. Pursuit of these aims will further advance our knowledge of the molecular and cellular events that underlie normal cardiac development, and will lead to new insights in the processes that can cause congenital heart disease and the role of the cushion tissues therein.